Microbial reduction of {66 {11 steroids

ABSTRACT

Delta 5-Steroids are reduced to the corresponding 5,6-dihydro5 Beta compounds in very high yield by using the organism Eubacterium nova. Among the products are coprosterol, 24-methyl5 Beta -cholestan-3 Beta -o1, and 24-ethyl-5 Beta -cholestan-3 Beta -o1.

United States Patent Eyssen Feb. 8, 1972 [54] MICROBHAL REDUCTlON OF ASTEROIDS [72] Inventor: Hendrik Eyssen, Holsbeek, Belgium [73] Assignee:Recherche et lndustrie Therapeutiques,

' R.I.T., Genval, Belgium 221 Filed: Nov. 26, 1969 [211 Appl.No.:880,400

[30] Foreign Application Priority Data Sept. 12, 1969 Great Britain..45,2l7/69 [52] U.S.CI. ..l95/51 R OTHER PUBLICATIONS PrimaryExaminer-Alvin E. Tanenholtz Attomey-William H. Edgerton, Richard D.Foggio, Joan S. Keps, Alan D. Lourie and Joseph A. Mariino [57] ABSTRACTA -Steroids are reduced to the corresponding 5,6-dihydro-5B compounds invery high yield by using the organism Eubacterium nova. Among theproducts are coprosterol, 24-methyl- 5B-cholestan-3B-ol and24-ethyl-5B-cholestan-3B-ol.

8 Claims, No Drawings MICROBIAL REDUCTION OF A STEROIDS The presentinvention relates to a process for the fermentative hydrogenation of Asteroids to yield 5,6-dihydro-5 B products.

The process of the present invention comprises subjecting a A steroid tothe reducing action of an Eubacterium nova species herein referred to byits ATCC No. 21,408 and isolating the resulting 5,6-dihydro-5 B product.

A certain number of 5,6-dihydro-5- B steroids are known and not all arenatural products.

Those who are not easily or not at all disponible as such from naturalsources are generally obtained by chemical modification of closelyrelated natural steroids already presenting the 5 ,6-dihydro-5 Bcharacteristics.

The attempts to obtain 5,6-dihydro-5 B steroids from the corresponding Aproducts by either chemical or microbiological methods were up to nownot encouraging because of the very limited yields of the knownprocesses or because of the further steps these processes necessitatefor isolating and purifying the obtained 5 B products.

So, the known chemical method consists in hydrogenating A steroids butthis reaction substantially affords 5,6-dihydro- 5-0: products with verylimited amounts of the 5 B compounds.

As to the known microbiological methods, they are based on the discoverythat nonidentified micro-organisms present in the intestine ofmammalians are responsible for the in situ hydrogenation of A steroidsto yield corresponding 5 B compounds which are thus possibly obtainedeither by extraction from feces or by unidentified micro-organismsmixtures cultivated from feces.

The instant Eubacterium ATCC No. 21,408 fermentation of A steroidspermits the hydrogenation of said A steroids to yield in substantiallystoechiometric amount the desired 5,6- dihydro-S B steroid which is theneasily isolated as a pure compound from the reaction medium.

Thus, it is the first object of the present invention to provide anEubacterium sp. fermentation method of A steroids and a further objectof the invention is the provision of a process for the production of5,6-dihydro-5 B steroids.

The instant process is useful to produce directly appreciable amounts ofpure 5,6-dihydro-5 B steroids the ones being useful as choleretic orhypocholesterolemic agents, while the other are useful as intermediatesfor the preparation of steroids.

The Eubacterium ATCC No. 21 ,408 is an Eubacterium nova species isolatedfrom the intestinal content of rats.

The strain presents straight or slightly curved small rods (the averagesize of which is 0.5 by 2 microns) occuring singly or in pairs,Gram-positive in young cultures, becoming Gramnegative after 3 to 5days.

The physiology examination reveals that the micro-organism grows bestfrom large inocula and, being strictly anaerobic, requires media withlow redox potentials. The optimal growth temperature is between 35 and42 C. The micro-organism does not grow or grows only very poorly onstandard bacteriological media unless supplemented with a A steroid(e.g., cholesterol).

Culture of Eubacterium ATCC No. 21,408 for the purpose and practice ofthe present invention is in or on a medium favorable to the developmentof this micro-organism but liquid media are preferred. Therefore, anystandard bacteriological liquid medium-provided it is supplemented withthe adequate A steroid-is suitable for the practice of the inventionwhen incubated anaerobically.

Examples of such media are fluid thioglycolate medium, e.g., from BBL(BBL Div. of BIOQUEST, Cockeysville, Md. U.S.A.) No. Ol397; cooked meatmedium, e.g., from BBL No. 01-658; trypticase soy broth, e.g., from BBLNo. 01-162. It has been found that growth on those media is stimulatedby addition of powdered animal tissue, such as for instance 5 percentfr'ceze dried meat powder or 5 percent freeze-dried brain powder.

The duration of the fermentation reaction is depending of differentconditions as, for instance, the age of the inoculum and its volume vs.the volume of the culture medium; the best inocula are those preparedfrom cultures at the end of the logarithmic phase of development. It hasbeen noticed that a complete transformation can be reached after a 24hours reaction period but prolongation of the fermentation for 10 daysdid not appear detrimental for the production of 5,6-dihydro- 5 Bsteroids.

Suitable A steroids for the practice of the present invention are any Asteroids not otherwise substituted in position 3 than by an oxo orhydroxyl group. In case of 3-keto A steroids, there is obtained amixture of the corresponding 3-011-5 B compound and of the 3-keto-5 Bcompound.

The following A steroids are given as possible starting materials:

1. Those presenting the A -androstene structure and, among them:

A -androstene derivatives such as A -androstene-3 B,

diol; A -androstene-3 B, 17 B-diol (androstenediol); A androsten -3B-ol-l7-one (dehydroepiandrosterone); l7a-methyl-5-androstene-3B,17B-dial (methandriol) and 17B [3-(dimethylamino)propyl] methylamino}-androst-S -en-3B-ol (azacosterol 2. Those presenting the A -pregnestructure and, among them:

A -pregnene derivatives suc h as A -pregnen-3 Bol-20-one (pregnenolone)21-acyloxy derivatives (e.g., 2l-acetoxypregnenolone), A -pregnene-3B,ZOB-diol and A pregnene-3B, 20a-diol.

3. Those presenting the A -cholestene structure and, among them:

A -cholestene itself and A -cholestene derivatives such as 5-cholesten-3-one, cholesterol and epicholesterol; cholestadienederivatives, e.g., cho1esta-5:6,7:8-dien-3-o1 (7-dehydrocholesterol) andcholesta-S16,24225-dien-3-ol (desmosterol).

4. A -campestene derivative-s e.g., campest-5-en-3B-ol (campesterol).

5. Those presenting the A -ergostene structure and, among them:

ergostatriene derivatives such as ergosterol, 9B-ergosterol, lumisteroland 9a-lumisterol and derivatives thereof (e.g.,22,23-dihydroergosterol); ergostatetraene derivatives such asdehydroergosterol.

6. Those presenting the A -stigmastene structure and, among them:

A -stigmastene derivatives such as sgigmast-5-en-3B-ol (B- sitosterol);stigmastadiene derivatives such as stigmasta- 5:6,22:23-dien 3-ol(stigmasterol) and 7- dehydrositosterol.

7. A -spirostene derivatives such as ruscogenin, diosgenin,

neoruscogenin, gentrogenin, rubijervine, isorubijervine and solasodine.

The following examples are illustrative of the process of the presentinvention and are not to be construed as limiting the invention.

EXAMPLE 1 Freeze-dried beef brain powder is extracted by acetone and 20g. of the dried residue is suspended in a medium consisting ofTrypticase peptone (a product from BBL Div. of Bioquest,

and a suspension of 7 g. of cholesterol (5-cholesten-3B-ol) and 5 g. oflecithin in 200 ml. of water is added thereto.

The mixture is sterilized by heating for 20 minutes at 120 C., cooledand inoculated with 10 ml. of a culture of Eubacterium ATCC No. 21,408prepared by inoculation of 1 ml. of the original culture in 10 ml. ofthe above medium and incubation for days in the absence of oxygen.

The medium is then incubated anaerobically for days at 37 C.

After that reaction time, the reaction mixture is extracted by four 700ml. portions of petroleum ether (B.P. 40-60 C.)

which are then pooled, washed with water, dried over sodium sulfate,filtered and the filtrate is evaporated under reduced pressure.

The oily residue is taken up in 400 ml. of potassium hydroxide (10percent) in ethanol and hydrolyzed by refluxing for 4 hours.

After cooling and addition of 400 ml. of water, the solution isextracted by four 400 ml. portions of petroleum ether (B.P. 40-60 C.)which are then pooled and washed by four 250 m1. portions of water toreach neutral pH of the washings.

The organic solution is dried over sodium sulfate, evaporated todryness, taken up again in 50 ml. of petroleum ether and chromatographedon 200 g. of Silica Gel for column chromatography(a product from E.Merck, Darmstadt, Germany) in a column of 34 mm. in diameter.Chromatography is carried out by stepwise elution with increasingconcentrations of ethylacetate in petroleum ether: 500 ml. of 1 percentethylacetate, 500 ml. of 2 percent ethylacetate, 500 ml. of 4 percentethylacetate, 500 ml. of 6 percent ethylacetate and 200 m1. of 8 percentethylacetate, successively.

Coprosterol is eluted with the 8 percent ethylacetate fraction andemerges in the effluent well before cholesterol.

The coprosterol-containing fraction is evaporated under reduced pressureand the residue is taken up in acetone from which 6.175 g. ofcoprosterol crystallize, showing the following characteristics:

one spot after thin layer chromatography on silica gel plates withpetroleum ether-ethylacetate, 9:1. In this system the substance showsthe same Rf-value (relative to reference cholesterol) as referencecoprosterol (1.44) and is distinctly separated from cholesterol (1.00),cholestanol (0.90) and epicoprostanol (1.24).

by gas chromatography on a 120 cm. column of 1 percent .1 XR on GasChrom Q (JXR on Gas Chrom Q is a product from Applied Science Labs,State College, Pa., U.S.A.) 100-120 mesh, only one peak is detected. Theretention time, relative to reference Sa-cholestan, corresponds toreference coprostanol (1.28) and is distinctly different fromcholesterol (1.34), cholestanol (1.34), 5/3- cholestan-3-one (1.34),5a-cholestan-3-one (1.41 4- cholesten-3-one( 1.54) and 5-cholesten-3-one(1.75).

by gas chromatography on a 5-foot column of 3 percent QF-l on Gas ChromQ (QF-l on Gas Chrom Q is a product from Applied Science Labs, StateCollege, Pa, U.S.A,) 100-120 mesh, only one peak is detected. Theretention time (relative to reference Sa-cholestan) is corresponding tothat of reference coprosterol (2.03), and is distinctly different fromreference 5/3-cholestan-3a-ol (2.26) and cholesterol (2.26).

by mass spectrography, the fractionation pattern is identical to that ofreference coprosterol and the molecular weight is 388.

EXAMPLE 2 Freeze-dried beef brain powder is extracted by acetone and 20g. of the dried residue is suspended in a medium consisting ofTrypticase peptone (a product from BBL Div. of Bioquest,

Sodium sulfite 0.168 Agar 0.42 5. Water 500 m1.

and a suspension of 7 g. of 24-methyl-5-cho1esten-3fi-ol (campesterol)and 5 g. of lecithin in 200 ml. of water is added thereto.

The mixture is sterilized by heating for 20 minutes at 120 C., cooledand inoculated with 10 ml. of a culture of Eubacterium ATCC No. 21,408prepared by inoculation of 1 ml. of the original culture in 10 ml. ofthe above medium and incubation for 5 days in the absence of oxygen.

The medium is then incubated anaerobically for 10 days at 37 C.

After that reaction time, the reaction mixture is extracted by four 700ml. portions of petroleum ether (B.P. 40-60 C.) which are then pooled,washed with water, dried over sodium sulfate, filtered and the filtrateis evaporated under reduced pressure.

The oily residue is taken up in 400 ml. of potassium hydroxide (10percent) in ethanol and hydrolyzed by refluxing for 4 hours.

After cooling and addition of 400 ml. of water, the solution isextracted by four 400 ml. portions of petroleum ether (B.P. 40 -60 C.)which are then pooled and washed by four 250 ml. portions of water toreach neutral pH of the washings.

The organic solution is dried over sodium sulfate, evaporated todryness, taken up again in 50 ml. of petroleum ether and.chromatographed on 200 g. of Silica Gel for column chromatography (aproduct from E. Merck, Darmstadt, Germany) in a column of 34 mm.diameter. Chromatography is carried out by stepwise elution withincreasing concentrations of ethylacetate in petroleum ether: 500 ml. of1 percent ethylacetate, 500 ml. of 2 percent ethylacetate, 500 m1. of 4percent ethylacetate, 500 m1. of 6 percent ethylacetate and 200 ml. of 8percent ethylacetate, successive- The reaction product is eluted withthe 8 percent ethylacetate fraction and emerges in the effluent wellbefore campesterol.

The reaction product-containing fraction is evaporated under reducedpressure and the residue is taken up in acetone from which 6.15 g. of24-methyl-5 B-cholestan-3 B-ol crystallize, showing the followingcharacteristics:

one spot on thin layer chromatography on silica gel plates withpetroleum ether-ethylacetate, 9:1. In this system the substance showsthe same Rf-value (relative to that of cholesterol)as reference24-methyl-5Bcholestane-38-01 (1.43), and is distinctly separated from24-methyl-5- cholestene-3B-ol 1.00).

by gas chromatography on a 120 cm. column of 1 percent JXR on Gas ChromQ (JXR on Gas Chrom Q is a product from Applied Science Labs, StateCollege, Pa, U.S.A.) 100-200 mesh, only one peak is detected. Therelative retention time (relative to reference Sacholestan) correspondsto that of reference 24-methyl-5 Bcho1estan-3 B- 01 (1.46), and isdistinctly different from that of 24- methyl-5-cholesten-3l3ol (1.54),24-ethyl-5-cholesten-3 13-01 (1 .70), and 24-ethyl-5Bcholesten-3B-ol1.64).

by mass spectography the fractionation pattern is identical to that ofreference 24-methy1-5Bstigmastan-3B-ol, and the molecular weight is 402.

EXAMPLE 3 Freeze-dried beef brain powder is extracted by acetone and 20g. of the dried residue is suspended in a medium consisting ofTrypticase peptone (a product from Agar Water and a suspension of 7 g.of 24-ethyl-5-cholesten-3fi-ol and g. of lecithin in 200 ml. of water isadded thereto.

The mixture is sterilized by heating for 20 minutes at 120 C., cooledand inoculated with ml. of a culture of Eubacterium ATCC No. 21,408prepared by inoculation of 1 ml. of the original culture in 10 ml. ofthe above medium and incubation for 5 days in the absence of oxygen.

The medium is then incubated anaerobically for 10 days at 37 C.

After that reaction time, the reaction mixture is extracted by four 700ml. portions of petroleum ether (B.P. 4060 C.) which are then pooled,washed with water, dried over sodium sulfate, filtered and the filtrateis evaporated under reduced pressure.

The oily residue is taken up in 400 ml. of potassium hydroxide (10percent) in ethanol and hydrolyzed by refluxing for 4 hours.

After cooling and addition of 400 ml. of water, the solution isextracted by four 400 ml. portions of petroleum ether (B.P. 40-60 C.)which are then pooled and washed by four 250 ml. portions or water toreach neutral pH of the washings.

The organic solution is dried over sodium sulfate, evaporated todryness, taken up again in 50 m1. of petroleum ether and chromatographedon 200 g. of Silica Gel for column chromatography (a product from E.Merck, Darmstadt, Germany) in a column of 34 mm. diameter.Chromatography is carried out by stepwise elution with increasingconcentrations of ethylacetate in petroleum ether: 500 ml. of 1 percentethylacetate, 500 ml. of 2 percent ethylacetate, 500 ml. or" 4 percentethylacetate, 500 ml. of 6 percent ethylacetate and 200 ml. of 8 percentethylacetate, successively.

The reaction product is eluted with the 8 percent ethylacetate fractionand emerges in the effluent well before 24-ethyl-5-cholesten-3[3-ol.

The reaction product-containing fraction is evaporated under reducedpressure and the residue is taken up in acetone from which 5.67 g. of24-ethyl-5B-cholestan-3B-ol crystallize, showing the followingcharacteristics:

one spot after thin layer chromatography on silica gel plates withpetroleum ether-ethylacetate, 9:1. In this system the substance showsthe same Rf-value as reference 24-ethyl- SB-cholestan-BB-ol (1.43 )andis distinctly separated from reference 24-ethyl-5-cholesten-3B-ol(1.00).

by gas chromatography on a 120 cm. column of 1 percent J XR on Gas ChromQ (JXR on Gas Chrom Q is a product from Applied Science Labs, StateCollege, Pa, U.S.A.) 100-120 mesh, only one peak can be detected. Therelative retention time (relative to reference Sa-cholestan) correspondsto that of reference 24-ethyl-5B-cholestan- 35-01 (1.64), and isdistinctly different from that of 24- ethyl-5-cholesten-3B-ol (1.70),24-methyl-5-cho1esten-3 3-01 (1.54), 24-methyl-5B-cholestan-3fl-ol(1.46), 24- ethyl-S,22-cholestadien-3B-ol (1.60), and 24-ethyl-5B-22-cholesten-3/3-ol l .55).

by mass spectrogramphy the fractionation pattern is identical with thatof reference 24-ethyl-5B-cholestan-3B-ol, and the molecular weight is416.

EXAMPLE 4 Freeze-dried beef brain powder is extracted by acetone and g.of the dried residue is suspended in a medium consisting of Trypticasepeptone (a product from BBL Div. of Bioquest,

Water 500 m1.

and a suspension of 7 g. of 24-ehty1-5,22-cho1estadien-3B-ol(stigmasterol) and 5 g. of lecithin in 200 ml. of water is addedthereto.

The mixture is sterilized by heating for 20 minutes at C., cooled andinoculated with 10 ml. of a culture of Eubacterium ATCC No. 21,408prepared by inoculation of 1 ml. of the original culture in 10 ml. ofthe above medium and incubation for 5 days in the absence of oxygen.

The medium is then incubated anaerobically for 10 days at 37 C.

After that reaction time, the reaction mixture is extracted by four 700ml. portions of petroleum ether (B.P. 4060 C.) which are then pooled,washed with water, dried over sodium sulfate, filtered and the filtrateis evaporated under reduced pressure.

The oily residue is taken up in 400 ml. of potassium hydroxide (10percent) in ethanol and hydrolyzed by refluxing for 4 hours.

After cooling and addition of 400 ml. of water, the solution isextracted by four 400 ml. portions of petroleum ether (8.1. 40 60 C.)which are then pooled and washed by four 250 ml. portions of water toreach neutral pH of the washings.

The organic solution is dried over sodium sulfate, evaporated todryness, taken up again in 50 ml. of petroleum ether and chromatographedon 200 g. of Silica Gel for column chromatography (a product from E.Merck, Darmstadt, Germany) in a column of 34 mm. diameter.Chromatography is carried out by stepwise elution with increasingconcentrations of ethylacetate in petroleum ether: 500 ml. of 1 percentethylacetate, 500 ml. of 2 percent ethylacetate, 500 ml. of 4 percentethylacetate, 500 ml. of 6 percent ethylacetate and 200 ml. of 8 percentethylacetate, successively.

The reaction product is eluted with the 8 percent ethylacetate fractionand emerges in the effluent well before stigmasterol.

The reaction product-containing fraction is evaporated under reducedpressure and the residue is taken up in acetone from which 5.15 g. of24-ethyl-5B-22-cho1esten-3B-olcrystallize, showing the followingcharacteristics:

by thin layer chromatography on silica gel plates with petroleumether-ethylacetate, 9:1, only one spot is detected. The Rf-value of thisspot relative to cholesterol corresponds to that of reference24-ethyl-5B-22- cholesten-3B-o1 (1.46) and is distinctly separated from24-ethyl-5 ,22-cholestadien-3B-ol 1.00).

by gas chromatography on a 120 cm. column of 1 percent JXR on Gas ChromQ (JXR on Gas Chrom Q is a product from Applied Science Labs, StateCollege, Pa, U.S.A.) only one peak is observed, and the retention time(relative to Sa-cholestan) corresponds to that of reference 24-ethyl-5B-22-cholesten-3B-ol (l.55)and is distinctly different from theretention times of 24-ethyl-5,22- cholestadien-3B-ol (1.60),24-ethyl-5B-cholestan-3,6-ol (1.64), 24-ethyl-5-cholesten-3B-ol (1.70),and 24- methyl-SB-cholestan-3B-ol l .46).

by mass spectrography, the fractionation pattern is identical with thatof reference 24-ethyl-5B-cholestan-33-01, and the molecular weight is414.

EXAMPLE 5 S-Pregnen-Elfi, 20B-diol (15 mg. and lecithin (40 mg. aresuspended in 1 ml. of water and the suspension is poured into 10 ml. ofa medium consisting of:

Trypticase peptone (a product from BBL Div. of Bioquest,

Agar 0.42 g. Yeast extract 7 g. Acetone extracted beef brain 20 g. Water700 ml.

The mixture is sterilized by heating for 20 minutes at 120 C., cooledand inoculated with 10 ml. of a culture of Eubacterium ATCC No. 21,408prepared by inoculation of 1 ml. of the original culture in 10 ml. ofthe above medium and incubation for 5 days in the absence of oxygen.

The medium is then incubated anaerobically for 7 days at 37 C.

After fermentation the medium is saponified with 30 ml. of 5 percent KOHin ethanol at refluxing temperature for 2 hours.

After cooling and addition of 30 ml. of water, the un-. saponifiablefraction is extracted three times with an equal volume of diethyl ether.The ether layers are pooled, washed with water to neutrality, dried overNa SO and filtered to yield 12.75 mg. of 5B-pregnan-3B, 20,8-dil showingthe following characteristics:

by thin layer chromatography of the ether extracts on silica gel usingpetroleum ether-ethylacetate, 7:3, only one spot is detected. TheRf-value of this spot relative to cholesterol corresponds to that ofreference SB-pregnan- 3B, 20/3-diol (0.41), and is distinctly differentfrom that of a-pregnan-3B, 20B-diol (0.34), 5-pregnen-3B, 20B- diol(0.34) and cholesterol (1.00).

by gas chromatography on a 120 cm. column of 1 percent JXR on Gas ChromQ (JXR on Gas Chrom Q is a product from Applied Science Labs, StateCollege, Pa, U.S.A.) 100-120 mesh, the retention time (relative to5acholestan) of the transformation productcorresponds to that ofreference 5B-pregnan-3B, 20B-diol (0.72) and is distinctly differentfrom that of 5a-pregnan-3B, ZOB-diol (0.76) and that of S-pregnen-SB,ZOB-diol (0.76).

by mass spectrography, the fractionation pattern is identical to that ofreference 5B-pregnane-3B, 20B-diol and the molecular weight is 320.

EXAMPLE 6 5-Androsten-3B-ol-17-one (dehydroepiandrosterone) 15 mg.) andlecithin (40 mg.) are suspended in 1 m1. of water and the suspension ispoured into 10 m1. of a medium consisting of:

Trypticase peptone (a product from BBL Div. of Bioquest,

The mixture is sterilized by heating for 20 minutes at 120 C., cooledand inoculated with 10 ml. of a culture of Eubacterium ATCC No. 21,408prepared by innoculation of 1 ml. of the original culture in 10 ml. ofthe above medium and incubation for 5 days in the absence of oxygen.

The medium is then incubated anaerobically for 7 days at 37 C.

After fermentation the medium is saponified with 30 ml. of 5 percent KOHin ethanol at refluxing temperature for 2 hours.

After cooling and addition of 30 ml. of water, the unsaponifiablefraction is extracted three times with an equal volume of diethyl ether.The ether layers are pooled, washed with water to neutrality, dried overNa SQ, and filtered to yield 12 mg. of 5B-androstan-3B-ol-17-one showingthe following characteristics:

by thin layer chromatography of the ether extracts on silica gel usingpetroleum ether-ethylacetate, 7:3, only one spot is detected. TheRf-value of this spot relative to cholesterol corresponds to that ofSB-androstan-SB-ol- 17-one (0.585) and is distinctly different from thatof 5aandrostan-3B-o1-17-one (0.44), 5-androsten-3B-ol-l7- one (0.50) andcholesterol (1.00).

by gas chromatography on a 120 cm. column of l percent JXR on Gas ChromQ (JXR on Gas Chrom Q is a product from Applied Science Labs, StateCollege, Pa, USA.) -120 mesh, the retention time (relative toSatcholestan) of the transformation product corresponds to that of5B-androstan-3/3-ol-l7-one (0.54), and is distinctly different from thatof 5a-androstan-3B-o1-17- one (0.57), and 5-androsten-3B-o1-17-one(0.57).

by mass spectrography the fractionation pattern is identical to tat ofreference SB-androstan-SB-ol-17-one, and the molecular weight is 290.

EXAMPLE 7 The technique is that described in Example 5 but, instead ofemploying 5-pregene-3B, 20B-diol, there is employed A -anmethylamino-androst-5-en-3B-ol (azacosterol); A pregnen-3B-o1-20-one(pregnenolone); 5-pregnene3B, 21- diol20-one-21-acetate(21-acetoxypregnenolone); 5 pregnene-3/3, 20a-dio1; A -cholestene; A-cholesten-3-one; 5- cholesten -3a-ol (epicholesterol);cholesta-S:6,7:8-dien-3-ol (7-dehydro-cho1esterol); cho1esta-5:6,24:25-dien-3ol (desmosterol); ergosta-5,7,22-trien-3B-o1(ergosterol); 98ergosta-5,7,22-trien-3B-ol (QB-ergosterol); 9B,l0aergosta- 5,7,22-trien-3Bo1 (lumisterol); 9a,10a-ergosta-5,7,22-trien- 3301 (9rx-lumisterol); ergosta-5,7-dien-3B-ol(22,23-dihydroergosterol); ergosta-5,7,9(1l),22 tetraen -3B-o1(dehydroergosterol); stigmasta-5,7 dien-Sfl-ol (7-dehydro-sitosterol); AZOB 2211p, 250: spirostene-lfi, 3B-diol (ruscogenin); 13 -20 B 2211 25a-spirosten -3B-ol (diosgenin); 18 -2013,, 2201 25a spirostene-lB,3B-diol-25 L (neoruscogenin); A 2013,, 22(1 2Sa -spirosten-3B-ol, 12 one(gentrogenin); A -solanidene-3B, 12a-diol (rubijervine); A-solanidene-3B, 18-diol (isorubijervine); A -20B,.-, 2201 25(1p, 27azaspirosten -33 01(solasodine); to yield 5/3-androstane-3B, 16a-dio1;5B-androstane-3B, 17B-diol; 17a-methyl-5B-androstane-3B, 17B-diol; 17B-[[3-dimethylamino)propyl]methylamino -5B-androstan-3B-ol; pregnan-3B-o1-20-one (3B-hydroxy-SB-pregnan-ZO-one); 21-acetoxy- SB-pregnan-3B-o1-20'one; 5/3-pregnane-3B, ZOa-diol; 5B- cholestane (coprostane);5/3-cholestan-3-one and 5B- cholestan-3B-ol; 5B-cho1estan-3a-ol;5B-cho1esta-7z8-en-3B- o1; 5B-cho1esta-24z25-en-3B-ol;5B-ergosta-7,22-dien-9fl-ol; 5B, 9B-ergosta-7,22-dien-3B-ol; 5B, 9B,10oz-ergosta-7.22- dien-3B-o1; 513, 9a, 10a-ergosta-7,22-dien-3B-o1;5B-ergosta-7 -en-3B-o1; 5B-ergosta-7,9(1 1), 22 trien-3B-ol;5B-stigmast-7- en-3B-ol; 58, 2013 22o 25a -spirostane-1B, 3B-diol; 5B,2013 22a 25 -spirostan-3B-ol; 53, 2013 2211p, 25a,.--spirostan-3B-ol-25L; 58, 206p, 2211p, 25a -spirostan-3B-ol-12 one;SB-solanidane-BB, 12a-diol; 5/3-so1anidane-3B, l8-diol; 5B, 208;, 22Clp,2501 27-azaspirostan-3/3-ol, respectively.

Whatl claim is:

1. A method of producing a SB-steroid which comprises subjecting a 3-oxoor 3-hydroxy- A steroid to microbiological reduction with enzymes ofEubacterium ATCC No. 21,408 and recovering the resulting 5,6-dihydro-5Bproduct.

2. A method of producing a 3-Ol-l-5B-steroid according to claim 1 whichcomprises subjecting a 3-keto A steroid to microbiological reductionwith enzymes of Eubacterium ATCC No. 21,408 and recovering the resulting5,6-dihydro- 513 -product.

3. A method of producing coprosterol according to claim 1 whichcomprises subjecting cholesterol to microbiological reduction withenzymes of Eubacterium ATCC No. 21,408 and recovering the resultingcoprosterol.

ATCC No. 21,408 and recovering the resulting 24-ethyl-5B-22-cholesten-3B-ol.

7. A method of producing 5B-pregnane-3B, 2OB-diol according to claim 1which comprises subjecting S-pregnene-Il/i, ZOB-diol to microbiologicalreduction with enzymes of Eubacterium ATCC No. 21,408 and recovering theresulting 5B- pregnane-3B, 20B-diol.

8. A method of producing SB-andr0stan-3B-ol-l7'one according to claim 1which comprises subjecting dehydro-epiandrosterone to microbiologicalreduction with enzymes of Eubacterium ATCC No. 21,408 and recovering theresulting 5B- androstan-3 B-oll 7-one.

2. A method of producing a 3-OH-5 Beta -steroid according to claim 1which comprises subjecting a 3-keto Delta 5 steroid to microbiologicalreduction with enzymes of Eubacterium ATCC No. 21,408 and recovering theresulting 5,6-dihydro-5 Beta -product.
 3. A method of producingcoprosterol according to claim 1 which comprises subjecting cholesterolto microbiological reduction with enzymes of Eubacterium ATCC No. 21,408and recovering the resulting coprosterol.
 4. A method of producing24-methyl-5 Beta -cholestan-3 Beta -ol according to claim 1 whichcomprises subjecting campesterol to microbiological reduction withenzymes of Eubacterium ATCC No. 21,408 and recovering the resulting24-methyl-5 Beta -cholestan-3 Beta -ol.
 5. A method of producing24-ethyl-5 Beta -cholestan-3 Beta -ol according to claim 1 whichcomprises subjecting Beta -sitosterol to microbiological reduction withenzymes of Eubacterium ATCC No. 21,408 and recovering the resulting24-ethyl-5 Beta -cholestan-3 Beta -ol.
 6. A method of producing24-ethyl-5 Beta -22-cholesten-3 Beta -ol according to claim 1 whichcomprises subjecting stigmasterol to microbiological reduction withenzymes of Eubacterium ATCC No. 21,408 and recovering the resulting24-ethyl-5 Beta -22-cholesten-3 Beta -ol.
 7. A method of producing 5Beta -pregnane-3 Beta , 20 Beta -diol according to claim 1 whichcomprises subjecting 5-pregnene-3 Beta , 20 Beta -diol tomicrobiological reduction with enzymes of Eubacterium ATCC No. 21,408and recovering the resulting 5 Beta -pregnane-3 Beta , 20 Beta -diol. 8.A method of producing 5 Beta -androstan-3 Beta -ol-17-one according toclaim 1 which comprises subjecting dehydro-epiandrosterone tomicrobiological reduction with enzymes of Eubacterium ATCC No. 21,408and recovering the resulting 5 Beta -androstan-3 Beta -ol-17-one.